1. Technical Field
The present invention relates to the field of cell culture technology and relates to methods of replicating/cloning cells, preferably cell lines which are important for the production of biopharmaceuticals. The invention also relates to methods of preparing proteins using cells that have been obtained and replicated and compositions which makes it possible to replicate individual cells.
2. Background
The market for biopharmaceuticals for treating humans is growing fast throughout the world. Over 900 biopharmaceuticals are currently being tested in clinical trials with an estimated potential turnover of 50 billion in 2010 (Datamonitor 2007). At present, an ever increasing number of biopharmaceuticals are produced in mammalian cells, as these have the capability for the correct processing and modification of a human protein. The successful production of high yields of biopharmaceuticals in mammalian cells is therefore crucial and depends on the characteristics of the recombinant monoclonal producing cell line that is used in the manufacturing process. In addition the duration of the cell line development is a critical time factor as to how quickly the biopharmaceutical can enter into clinical trials. In view of these aspects there is an urgent need to speed up the process of developing cell lines and make it more efficient.
For the biotechnological production of biologically active or therapeutic proteins in mammalian cells, so-called biopharmaceuticals, the corresponding mammalian cells are stably transfected with DNA which codes for the biologically active protein (or its subunits). After the transfection process a pool of millions of differently transfected cells is normally obtained. Therefore the crucial step for the preparation of efficient production cell lines is in the selection and replication of cell clones which on the one hand grow very stably and on the other hand show a high specific productivity of therapeutic protein (product formation etc.). As there are millions of different product-expressing cells, it is critical to be able to analyse a plurality of cells individually with a high throughput and using automation in order to be able to sort out suitable candidates (single cell clones) which both grow very robustly and also yield high product titters. This process of single cell isolation and subcultivation is known as cloning or recloning.
The use of animal cell cultures for producing biopharmaceuticals demands a genotypically and phenotypically homogeneous, i.e. monoclonal cell culture. This is achieved by recloning techniques such as “limited dilution” or by the automated depositing of individual cells by fluorescence activated cell sorting (FACS).
However, there is the problem of effectively replicating typical recombinant production cells such as mouse myeloma (NS0), hamster ovary (CHO), or hamster kidney cells (BHK), particularly if they are adapted to growth in serum-free suspension cultures, i.e. under modern production-relevant cell culture conditions, after recloning, whereby the cells are individually deposited in microtitre plates, under serum-free culture conditions.
The reason for this is that cells in vivo are embedded in the tissue matrix and are supplied with secreted auto- and paracrine growth factors by adjacent cells. They are therefore not adapted to isolated growth and die off without stimulation by growth factors if they are not slowly adapted to the new conditions.
In particular, the use of serum-free or chemically defined media in the recloning step leads to a restricted recloning efficiency, i.e. only a small percentage of the cells deposited survive and grow into a monoclonal cell line.
The “limited dilution” and FACS recloning techniques currently used are well known in the art.
In “limited dilution” the cell suspension is serially diluted and the cells are then deposited in a microtitre plate in different numbers of cells per well. In wells containing large numbers of cells, many or all the cells survive as the result of adequate secretion of autocrine growth factors. The fewer cells are seeded per well, the fewer cells survive, so that in this way the dilution can be adjusted so that statistically only one cell survives per well and grows into a monoclonal line. These individual cells clones are detected by visual and/or imaging techniques and the cell clones are grown on in larger culture vessels.
In FACS technology, a flow cytometer is used to generate single cell clones. For this the cells are placed in a laminar flow and are individually steered into the wells of the microtitre dishes. This ensures that the surviving colonies really are individual clones. Therefore, FACS technology is the preferred method compared with Limited Dilution.
The use of serum-free or chemically defined media in the recloning step leads to restricted recloning efficiency, i.e. only a few percent of the individually deposited cells grow into a monoclonal cell line.
At a low recloning efficiency, therefore, a number of microtitre plates have to be filled with single cells in order to obtain the desired number of individual clones, which is time-consuming and expensive (e.g. in terms of media, dishes, etc).
A low recloning efficiency is particularly disadvantageous if the subsequent analysis of the single cell clones is to be carried out using an automated system. A analysing robot cannot normally distinguish between wells containing living or dead cells and automatically measures all the wells in the microtitre plate. With a recloning efficiency of only 10%, this means that in 90% of cases the robot will analyse an empty well—and will use the same amount of reagent for this as for analysing a living cell clone. In the example here, therefore, 90% of the time and 90% of the material costs are wasted without any data being obtained.
To solve this problem, in the past, serum (e.g. foetal calf serum, FCS) has often been added to the medium. Serum contains an undefined mixture of different soluble proteins and growth factors which supports the survival and proliferation of cells. For regulatory reasons, however, the use of non-definable additions such as serum is increasingly less tolerated, partly because of the risk of infection with bovine viruses. Totally serum-free production of cell lines is therefore the state of the art from a regulatory point of view.
Another possible solution is to carry out a “limited dilution” for the recloning. As this method only leads statistically to the production of single cell clones but many clones may also grow in one well, this process has to be repeated several times (usually 2-3 times) to ensure that the cell line obtained really does originate from only a single clone. These repeated cycles involve high labour and time costs which have a negative effect on the costs and timelines required.
Another approach is the use of “feeder” cells. The name comes from the English word “feed” and refers to a co-cultivation with usually non-dividing cells which serve to supply the desired cells in the culture with nutrients and secreted growth factors. The recloning efficiency can be significantly increased by feeder cells.
In another approach, recombinant proteins are added to the recloning medium, to promote the survival and growth of the individually deposited cells. Examples of recombinant proteins used are insulin, insulin-like growth factor (IGF), epithelial growth factor (EGF) or human serum albumin (HSA). These additives are obtained as purified proteins or protein solutions and added to the medium. The disadvantages of this method are the costs, the dependency on the availability of the recombinant proteins and their instability. The disadvantages include, inter alia:                the need to store them at −70° C.,        freeze/thaw cycles lead to inactivation and therefore have to be avoided,        rapid loss of activity in solution under cultivation conditions.        
The aim of the present invention is to increase the recloning efficiency in the serum-free FACS-based cloning of production cells.